Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness

Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile. The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours. BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay. Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissue

Copy Number Variation at the FCGR Locus Includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B

Human Fc gamma receptors (Fc gamma Rs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low-affinity Fc gamma Rs are located on chromosome 1q23-24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter-individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23-24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low-affinity Fc gamma Rs in one assay, we have developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene-dosage effect of FCGR3A was found, that seemed to correlate nicely with the Fc gamma RIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co-segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with Fc gamma RIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B. (C) 2009 Wiley-Liss, In

Electrochemical Genetic Profiling of Single Cancer Cells

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

<div><p>Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of <i>EYS</i> revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in <i>RP1</i>, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.</p></div

Causative mutations and putatively pathogenic variants identified in this study.

<p>Causative alleles are being listed as “allele 1” and “allele 2” in resolved cases. Additional alleles are shown if the minor allele frequency is below 3% and if <i>in silico</i> prediction suggests putative pathogenicity. The inheritance pattern was largely delineated from pedigree informations. In patients 22, 23, 77, 100, 116 and 119, the true mode of inheritance had not been evident from the pedigree information and was finally deduced from the genotype. a, this study. References for studies cited in this table can be found in the Supplementary Material (References S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone.0078496.s001" target="_blank">File S1</a>). n.d., not defined; f, female; m, male; ar, autosomal recessive; ad, autosomal dominant; s, sporadic. Xl, X-linked. Cau, Caucasian; Ger, Germany; Tur, Turkey; KSA, Kingdom of Saudi Arabia; Pol, Poland; Au, Austria; Syr, Syria; Pak, Pakistan; DRC, Democratic Republic of the Congo; Mor, Morocco; UAE, United Arab Emirates; E-Eur, East Europe; SE-Eur, Southeast Europe.</p

Mutational spectrum in RP and LCA patients.

<p>Percentages refer to patients with mutations in the respective gene that are considered causative. The distribution of causative mutations across many genes, each contributing a relatively small fraction to the mutational spectrum, confirms the extensive genetic heterogeneity of retinal dystrophies. Note that the three patients that were found to carry X-linked mutations are not contained in the schemes A – B. <b>A.</b> arRP. <b>B.</b> adRP. Note that the percentages refer to a relatively small adRP cohort in this study. <b>C.</b> LCA. <b>D.</b> Functional categorization of genes that were found to carry causative mutations in our study. Mutations in genes encoding components of the photoreceptor’s connecting cilium and associated structures were predominant.</p

Comparison of this study with previous NGS studies on retinal dystrophies.

*<p>Positive controls not included.</p>**<p>Additional samples from the same families not included. Gene numbers in brackets include additionally screened candidate genes that are not yet proven retinal disease genes. BBS, Bardet-Biedl syndrome; CRD, cone-rod dystrophy; CD, cone dystrophy; CSNB, congenital stationary night blindness; FA, fundus albipunctatus; STGD, Morbus Stargardt; USH, Usher syndrome.</p

Hemizygosity of a <i>CRX</i> mutation in a recessive consanguineous LCA family.

<p><b>A.</b> Compound-heterozygosity for a potentially protein-extending no-stop mutation (c.899A>G/p.(*300Trpext*118); here designated as Ext) abrogating the natural termination codon in exon 4 and a deletion of the same exon (delE4) <i>in trans</i> in patient 110 and her brother. <b>B.</b> Graphical view of the LOD score calculation from genomewide SNP mapping for this family previous to NGS testing: Genomewide homozygosity mapping prior to NGS did not identify a clear candidate locus. The combined maximum parametric LOD score of 2.4 was not obtained. <b>C.</b> Scheme of the <i>CRX</i> gene and coverage plots for CNV analysis from NGS data (Illumina MiSeq), indicating a heterozygous deletion of exon 4 (upper panel, absolute coverage based on read count; lower panel, SeqNext CNV analysis). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone-0078496-g002" target="_blank">Figure 2C</a>. <b>D.</b> Schematic representation of the mapped sequencing reads for the no-stop mutation (Integrative Genomics Viewer). The mutation (arrow) was present in all 65 reads covering this region of the gene and therefore appeared homozygous. <b>E.</b> Electropherograms from Sanger sequencing of the no-stop mutation with hemizygosity in patient 110 (upper panel) and heterozygosity in her mother (lower panel). <b>F.</b> Summary of the disease-causing genetic constellation in patient 110 and her brother (superimposition on parental alleles).</p